organs affects mammary development. To this
end, we first analyzed coexpression of Fsp1 and
Gli2 in ovary, the major organ for female hormone production (e.g., estrogen and progesterone).
We observed few double-positive cells in corpus
luteum (0.16%, n = 1927), and no coexpression in
follicles (0%, n = 2418) (Fig. 4, A to D), suggesting
that Gli2 ablation is a rare event in the ovaries
of our Gli2DS mice. In addition, and consistent
with the lack of Fsp1 and Gli2 coexpression in
ovary, Gli2WT and Gli2DS mice had statistically
indistinguishable estrogen levels (Fig. 4E, P = 0.5)
that were comparable to those previously reported
(27). We similarly noted little coexpression of Fsp1
and Gli2 in the pituitary and no significant change
in serum growth hormone (GH) or prolactin levels (Fig. 4, F to H). These data collectively suggest
that the Gli2DS mice maintain a hormonal environment similar to that of Gli2WT littermates during development.

We next investigated potential coexpression of
Fsp1 and Gli2 in mammary macrophages, which
are reported to play a role in mammary development during puberty (26). Although we did observe Gli2 expression in a minuscule fraction (~1%)
of mammary cells expressing the macrophage
marker F4/80 (Fig. 5A, n = 1247), we detected no

Fsp1 expression in F4/80+ cells (Fig. 5B, n = 1248),
indicating that Fsp1Cre-driven ablation of Gli2 in
mammary macrophages is highly unlikely. To further exclude a possible defect in mammary macrophages in the Gli2DS phenotype, we transplanted
wild-type stromal cells or macrophages into the
mammary fat pads of pubertal Gli2DS;NSG recipient mice (Fig. 5C). We noted that although stromal cells successfully rescued ductal enlargement
with restoration of periductal collagen and extracellular matrix (Fig. 5, C to F, n = 3), macrophage
transplantation did not provide any rescuing activity. These results suggest that macrophages
contribute little if anything to the epithelial activity lost in Gli2DS mice. Furthermore, the sufficiency of locally implanted stromal cells to rescue
the Gli2DS mammary phenotype excludes a role for
loss of systemic factors such as pituitary or ovarian mammatropic hormones in the Gli2DS
mammary phenotype.

To identify stromal factors induced by Gli2activity, we compared gene expression of FACS-isolated Fsp1Cre-marked stromal cells from Gli2DSor Gli2WT littermates by microarray analysis) andidentified genes encoding paracrine factors suchas Igf1, Wnt2, Hgf, Fgf7, and Bmp7; we alsoidentified down-regulated genes encoding estrogenreceptor (Esr1), growth hormone receptor (Ghr),and other genes typically expressed in stromalcells. Of particular interest among the para-crine factors, WNT signals sustain the activity ofMaSCs in vivo and in ex vivo culture (28, 29), andfunctional inactivation of Igf1 or its epitheliallyexpressed receptor, Igf-1r, leads to development ofhypoplastic mammary glands with terminal endbud abnormalities (30, 31), similar to the defectswe observe in Gli2DS mice. The down-regulationof these genes was confirmed by qRT-PCR of bulkstromal cells from Gli2WT and Gli2DS mice (Fig. 6A)and was further quantified by qRT-PCR fromsingle cells (fig. S6; summarized in Fig. 6B). Igf1and Wnt2 were expressed in 7% and 9% of indi-vidual Gli2WT stromal cells, respectively, but in 0%of Gli2DS stromal cells (Fig. 6B). Our single-cellanalysis was validated by the expression of Gli2in 19% of Fsp1Cre-marked Gli2WT stromal cells (Fig.6B), consistent with our immunofluorescenceanalyses (Fig. 2, A and B). The Gli2nLacZ reporteris expressed within the thin stromal layer that re-mains in Gli2DS mice in a pattern similar to thatof Gli2nLacZ within control mice (Fig. 6C), indi-cating that these cells may be correctly specifiedbut fail to function normally, owing to loss of GLI2protein activity. These results collectively showZhao et al., Science 356, eaal3485 (2017) 21 April 2017 5 of 12

No significant difference
was found between wt
and Gli2DS mutant
groups (P = 0.5). (F)

Representative H&E
(left) immunostaining
(middle and right)
showing that Gli2-
expressing cells are distinct from Fsp1Cre-
labeled cells in the
anterior lobe of
pituitary gland in

Fsp1Cre;Rosa26mTmG/+;

Gli2nLacZ/+ mice. b-Gal (Gli2)–expressing cells were pseudocolored in red. The lack of Gli2 expression in the posterior lobe of the pituitary gland is apparent. (G and
H) Quantification of growth hormone and prolactin levels in serum by ELISA in Gli2WT or Gli2DS mice.